Our recent studies on human mammary cells have allowed us to identify a previously undetected phenotype in a rare subpopulation of human mammary epithelial cells (HMEC). As previously reported, the majority of epithelial cells that grow from biopsy tissue from healthy women respond to a proliferation barrier around 15 to 20 population doublings after placement in culture. After a transient arrest (called "selection" in culture), a rare subpopulation of cells (approximately 10-4 to 10-5) grows beyond the initial barrier and propagate for months in culture. These "post-selection" HMEC are typified by loss of specific cell cycle controls and the accumulation of a tremendous number of chromosomal abnormalities. As this population of cells is grown in culture, they approach a second growth plateau in which virtually 100% of the cells have chromosomal abnormalities. These observations challenge traditional views of how and when cells acquire genomic changes in cancer by providing a cell intrinsic mechanism that, early in the neoplastic process, generates multiple simultaneous genetic changes. These cells are generated without obligatory exposure to known physical, viral or chemical mutagenic agents. Finally, these cells possess defined characteristics that are often found in cancer cells and may explain their origin. "Post-selection" HMEC do not express p16, an important cyclin dependent kinase inhibitor, they lack proper checkpoint control and they do not maintain genomic integrity. Should these cells arise in vivo, they could represent the earliest steps in human mammary carcinogenesis. These observations also identify novel opportunities. They may provide potential markers for assessing susceptibility to neoplastic transformation in individuals as well as potential targets for prevention and therapy. Multiple markers clearly identify the different cellular states in vitro and have allowed for the identification of cells with these properties in vivo. Remarkably, the changes we detect in "post-selection" HMEC mimic many of the changes seen in premalignant lesions in breast cancer. We hypothesize that the above-described properties of "post-selection" HMEC in vitro are critically relevant to the transformation processes of mammary epithelial cells in vivo. The goals of this application are to (1) determine how p16 inactivation contributes to the "post-selection" HMEC phenotype, (2) determine the origins of "post-selection" HMEC, 3) determine if similar cells detected in vivo are (a) present in increased frequencies in individuals at high risk for breast cancer, and (b) exhibit characteristics of stem cells, and (4) examine selective cell cycle checkpoint controls in HMEC. These studies may provide novel targets for prevention or treatment of breast cancer.